Chemical structure of the lipid A component of Plesiomonas shigelloides and its taxonomical significance

Abstract The chemical structure of the lipid A moiety of the lipopolysaccharide of the type strain of Plesiomonas shigelloides was elucidated. It consists of a β-(1 → 6)-linked glucosamine disaccharide carrying phosphate groups at C-1 of the reducing and at C-4' of the non-reducing glucosamine. It contains a total of 6 residues of fatty acids, 2 amide-linked and 4 ester-linked. The amino groups of the backbone disaccharide are N -acylated by substituted 3-hydroxyacyl residues: at the reducing glucosamine by 3-O-(14:0)14:0; and at the non-reducing glucosamine by 3-O-(12:0)14:0.
Two residues of 3-hydroxytetradecanoic acid are linked to C-3 and C-3' of the glucosamine residues; the hydroxy groups of these ester-linked 3-hydroxytetradecanoic acids are unsubstituted. In free lipid A, the hydroxyl groups at C-4 and C-6' are unsubstituted, indicating that the 2-keto-3-deoxyoctonic acid (KDO) is linked to C-6' of the non-reducing glucosamine, as was shown with enterobacterial lipid A. The taxonomical significance of these structural details is discussed.     

Structure of Shigella sonnei lipid A

The detailed chemical structure of lipid A of Shigella sonnei phase II was elucidated. The lipid A backbone consists of a β-1,6-linked glucosamine disaccharide substituted with (mono) phosphates both at C-1 and C-4′. This was shown by selective degradation followed by ³¹P-NMR studies. C-4 and C-6′ were found to contain unsubstituted hydroxyl groups, the latter being the point of attachment of KDO as reported for other enterobacterial lipids A.The amino groups of the glucosamine disaccharide are substituted by 3-hydroxy fatty acids: 3-O-(14:0) 14:0 at the non-reducing glucosamine and 3-O-(12:0) 14:0 at the reducing glucosamine. In contrast to earlier reports, no ethanolamine or phosphodiester linkages were found in lipid A.     

Structural determination of lipid A of the lipopolysaccharide from Pseudomonas reactans. A pathogen of cultivated mushrooms.

The chemical structure of lipid A from the lipopolysaccharide of the mushroom-associated bacterium Pseudomonas reactans, a pathogen of cultivated mushroom, was elucidated by compositional analysis and spectroscopic methods (MALDI-TOF and two-dimensional NMR). The sugar backbone was composed of the beta-(1'-->6)-linked d-glucosamine disaccharide 1-phosphate. The lipid A fraction showed remarkable heterogeneity with respect to the fatty acid and phosphate composition. The major species are hexacylated and pentacylated lipid A, bearing the (R)-3-hydroxydodecanoic acid [C12:0 (3OH)] in amide linkage and a (R)-3-hydroxydecanoic [C10:0 (3OH)] in ester linkage while the secondary fatty acids are present as C12:0 and/or C12:0 (2-OH). A nonstoichiometric phosphate substitution at position C-4' of the distal 2-deoxy-2-amino-glucose was detected. Interestingly, the pentacyl lipid A is lacking a primary fatty acid, namely the C10:0 (3-OH) at position C-3'. The potential biological meaning of this peculiar lipid A is also discussed.     

Characterization of Mesorhizobium huakuii lipid A containing both D-galacturonic acid and phosphate residues.

The chemical structure of the free lipid A isolated from Mesorhizobium huakuii IFO 15243(T) was elucidated. Lipid A is a mixture of at least six species of molecules whose structures differ both in the phosphorylation of sugar backbone and in fatty acylation. The backbone consists of a beta (1'-->6) linked 2,3-diamino-2,3-dideoxyglucose (DAG) disaccharide that is partly substituted by phosphate at position 4'. The aglycon of the DAG-disaccharide has been identified as alpha-D-galacturonic acid. All lipid A species carry four amide-linked 3-hydroxyl fatty residues. Two of them have short hydrocarbon chains (i.e. 3-OH-i-13:0) while the other two have longer ones (i.e. 3-OH-20:0). Distribution of 3-hydroxyl fatty acids between the reducing and nonreducing DAG is symmetrical. The nonpolar as well as (omega-1) hydroxyl long chain fatty acids are components of acyloxyacyl moieties. Two acyloxyacyl residues occur exclusively in the nonreducing moiety of the sugar backbone but their distribution has not been established yet. The distal DAG amide-bound fatty acid hydroxyls are not stoichiometrically substituted by ester-linked acyl components.     

Heterogeneity of lipid A: structural determination by 13C and 31P NMR of lipid A fractions from lipopolysaccharide of Escherichia coli 0111

Purified lipid A from Escherichia coli 0111 was fractionated by thin-layer chromatography, and seven major bands were studied by 13C and 31P NMR. All lipid A fractions except one had fatty acids, 3-hydroxytetradecanoic acid, 3-(acyloxy)tetradecanoic acid, and phosphate groups bonded to the diglucosamine backbone. The remaining fraction was shown to be phosphatidylethanolamine. The number of substituents found showed that in all fractions all sites available for C-acylation (C-3, C-4, and C-3') and N-acylation (C-2 and C-2') carried acylic substituents. The number, ranging from four to six, and type of ester-bound carboxylic acid residues as well as the number of phosphate groups differed among the fractions. The three fastest moving bands all had three unsubstituted hydroxy fatty acids and one phosphate group (C-4'), while the slower moving bands had four hydroxy fatty acids and two phosphate groups. Unsubstituted 3-hydroxytetradecanoic acid residues were amide-bound to the disaccharide in all but one of the fractions. In summary, the heterogeneity of E. coli 0111 lipid A is found to be a consequence of a variation of the number and composition of carboxylic acid residues and of varying phosphate content.     

Complete structural characterization of the lipid A fraction of a clinical strain of B. cepacia genomovar I lipopolysaccharide

Burkholderia cepacia, a Gram-negative bacterium ubiquitous in the environment, is a plant pathogen causing soft rot of onions. This microorganism has recently emerged as a life-threatening multiresistant pathogen in cystic fibrosis patients. An important virulence factor of B. cepacia is the lipopolysaccharide (LPS) fraction. Clinical isolates and environmental strains possess LPS of high inflammatory nature, which induces a high level production of cytokines. For the first time, the complete structure of the lipid A components isolated from the lipopolysaccharide fraction of a clinical strain of B. cepacia is described. The structural studies carried out by selective chemical degradations, MS, and NMR spectroscopy revealed multiple species differing in the acylation and in the phosphorylation patterns. The highest mass species was identified as a penta-acylated tetrasaccharide backbone containing two phosphoryl-arabinosamine residues in addition to the archetypal glucosamine disaccharide [Arap4N-l-beta-1-P-4-beta-D-GlcpN-(1-6)-alpha-D-GlcpN-1-P-1-beta-L-Arap4N]. Lipid A fatty acids substitution was also deduced, with two 3-hydroxytetradecanoic acids 14:0 (3-OH) in ester linkage, and two 3-hydroxyhexadecanoic acids 16:0 (3-OH) in amide linkage, one of which was substituted by a secondary 14:0 residue at its C-3. Other lipid A species present in the mixture and exhibiting lower molecular weight lacked one or both beta-L-Arap4N residues.     

Occurrence of an Unusual Hopanoid-containing Lipid A Among Lipopolysaccharides from Bradyrhizobium Species

The chemical structures of the unusual hopanoid-containing lipid A samples of the lipopolysaccharides (LPS) from three strains of Bradyrhizobium (slow-growing rhizobia) have been established. They differed considerably from other Gram-negative bacteria in regards to the backbone structure, the number of ester-linked long chain hydroxylated fatty acids, as well as the presence of a tertiary residue that consisted of at least one molecule of carboxyl-bacteriohopanediol or its 2-methyl derivative. The structural details of this type of lipid A were established using one- and two-dimensional NMR spectroscopy, chemical composition analyses, and mass spectrometry techniques (electrospray ionization Fourier-transform ion cyclotron resonance mass spectrometry and MALDI-TOF-MS). In these lipid A samples the glucosamine disaccharide characteristic for enterobacterial lipid A was replaced by a 2,3-diamino-2,3-dideoxy-d-glucopyranosyl-(GlcpN3N) disaccharide, deprived of phosphate residues, and substituted by an α-d-Manp-(1→6)-α-d-Manp disaccharide substituting C-4′ of the non-reducing (distal) GlcpN3N, and one residue of galacturonic acid (d-GalpA) α-(1→1)-linked to the reducing (proximal) amino sugar residue. Amide-linked 12:0(3-OH) and 14:0(3-OH) were identified. Some hydroxy groups of these fatty acids were further esterified by long (ω-1)-hydroxylated fatty acids comprising 26–34 carbon atoms. As confirmed by mass spectrometry techniques, these long chain fatty acids could form two or three acyloxyacyl residues. The triterpenoid derivatives were identified as 34-carboxyl-bacteriohopane-32,33-diol and 34-carboxyl-2β-methyl-bacteriohopane-32,33-diol and were covalently linked to the (ω-1)-hydroxy group of very long chain fatty acid in bradyrhizobial lipid A. Bradyrhizobium japonicum possessed lipid A species with two hopanoid residues.     

Characterization of a novel lipid-A from Rhizobium species Sin-1. A unique lipid-A structure that is devoid of phosphate and has a glycosyl backbone consisting of glucosamine and 2-aminogluconic acid

The structure of the lipid-A from Rhizobium species Sin-1, a nitrogen-fixing Gram-negative bacterial symbiont of Sesbania, was determined by composition, nuclear magnetic resonance spectroscopic, and mass spectrometric analyses. The lipid-A preparation consisted of a mixture of structures due to differences in fatty acylation and in the glycosyl backbone. There were two different disaccharide backbones. One disaccharide consisted of a distal glucosaminosyl residue beta-linked to position 6 of a proximal 2-aminoglucono-1,5-lactonosyl residue, and in the second disaccharide, the proximal residue was 2-amino-2,3-dideoxy-d-erythro-hex-2-enono-1,5-lactone. For both disaccharides, the distal glucosamine was acylated at C-2' primarily with beta-hydroxypalmitate (beta-OHC16:0) which, in turn, was O-acylated with 27-hydroxyoctacosanoic acid. For some of the lipid-A molecules, the distal glucosaminosyl residue was also acylated at C-3' with beta-hydroxymyristate (beta-OHC14:0), whereas other molecules were devoid of this acyl substituent. Both the 2-aminoglucono-1,5-lactonosyl and 2-amino-2,3-dideoxy-d-erythro-hex-2-enono-1,5-lactonosyl residues were acylated at C-2, primarily with beta-OHC16:0. Minor amounts of lipid-A molecules contained beta-OHC14:0 at C-3 and/or beta-hydroxystearate (beta-OHC18:0) or beta-hydroxyoctadecenoate (beta-OHC18:1) as the C-2 and C-2' N-acyl substituents.     

Structural characterization of the lipid A component of pathogenic Neisseria meningitidis.

The lipid A component of meningococcal lipopolysaccharide was structurally characterized by using chemical modification methods, methylation analysis, 31P nuclear magnetic resonance, and laser desorption mass spectroscopy. It was shown that Neisseria meningitidis lipid A consists of a 1,4'-bisphosphorylated beta(1'----6)-linked D-glucosamine disaccharide (lipid A backbone), both phosphate groups being largely replaced by O-phosphorylethanolamine. This disaccharide harbors two nonsubstituted hydroxyl groups at positions 4 and 6', the latter representing the attachment site of the oligosaccharide portion in lipopolysaccharide. In addition, it is substituted by up to six fatty acid residues. In the major lipid A component, representing a hexaacyl species, the hydroxyl groups at positions 3 and 3' carry (R)-3-hydroxydodecanoic acid [12:0(3-OH)], whereas the amino groups at positions 2 and 2' are substituted by (R)-3-(dodecanoyloxy)tetradecanoic acid [3-O(12:0)-14:0]. A minor portion was present as a tetraacyl lipid A component lacking either dodecanoic acid (12:0) or 12:0 and 12:0(3-OH). N. meningitidis lipid A, therefore, significantly differs from Escherichia coli lipid A by the nature and locations of fatty acids and the substitution of O-phosphorylethanolamine for the nonglycosyl (4'-P) and glycosyl phosphate.     

Chemical studies on the lipopolysaccharide of Rhizobium meliloti 10406 and its lipid A region

The structure of the lipopolysaccharide from Rhizobium meliloti 10406, a derivative of the wild-type strain MVII-1, was examined. The compositional analysis of its polysaccharide moiety demonstrated lack of heptose(s), but high contents in glucose, galacturonic acid and 2-keto-3-deoxy-octonate (dOclA) as characteristic features. The lipid A moiety consisted of a -1,6 linked glucosamine disaccharide carrying ester (at C-4) and glycosidically (at C-1) linked phosphate residues, both present exclusively as monoester phosphates but not as phosphodiesters. Ester- and amidelinked 3-hydroxy fatty acids were mostly present as non-3-O-acylated residues. Laser desorption mass spectrometry (LD-MS) revealed heterogeneity in the fatty acid substitution, as was also indicated by the non-stoichiometric ratios obtained by quantitative fatty acid analysis. The predominating lipid A structure contained at the reducing glucosamine residue ester-linked 3-hydroxy-tetradecanoic acid (3-OH-14:0) and amide-linked 3-OH-18:0, or 3-OH-18:1, respectively. The distal (non-reducing) glucosamine carried ester-bound the recently discovered 27-hydroxyoctacosanoic acid and 3-OH-14:0 and, as amide-linked fatty acid, mostly 3-hydroxy-stearic acid (3-OH-18:0).The isolated lipopolysaccharide exhibited a high extent of lethal toxicity in galactosamine-treated mice, comparable to that of enterobacterial lipopolysaccharide. The structural relationship of LPS and lipid A of Rhizobium meliloti to other rhizobial lipopolysaccharides and lipid A's with respect to questions of taxonomy and of phylogenetic relationships will be discussed.Abbreviations LPS lipopolysaccharide - dOclA 3-deoxy-D-mannooctulosonic acid (KDO) - GalA galacturonic acid - DOC sodium deoxycholate - PAGE polyacrylamide gel electrophoresis - LD-MS laser desorption-mass spectrometry     

Structural analysis of the lipid A component of Campylobacter jejuni CCUG 10936 (serotype O:2) lipopolysaccharide. Description of a lipid A containing a hybrid backbone of 2-amino-2-deoxy-D-glucose and 2,3-diamino-2,3-dideoxy-D-glucose

The chemical structure of Campylobacter jejuni CCUG 10936 lipid A was elucidated. The hydrophilic backbone of the lipid A was shown to consist of three (1----6)-linked bisphosphorylated hexosamine disaccharides. Neglecting the phosphorylation pattern, a D-glucosamine (2-amino-2-deoxy-D-glucose) disaccharide [beta-D-glucosaminyl-(1----6)-D-glucosamine], a hybrid disaccharide of 2,3-diamino-2,3-dideoxy-D-glucose and D-glucosamine [2,3-diamino-2,3-dideoxy-beta-D-glucopyranosyl-(1----6)-D-glucosamine], and a 2,3-diamino-2,3-dideoxy-D-glucose disaccharide were present in a molar ratio of 1:6:1.2. Although the backbones are bisphosphorylated, heterogeneity exists in the substitution of the polar head groups. Phosphorylethanolamine is alpha-glycosidically bound to the reducing sugar residue of the backbone, though C-1 is also non-stoichiometrically substituted by diphosphorylethanolamine. Position 4' of the non-reducing sugar residue carries an ester-bound phosphate group or is non-stoichiometrically substituted by diphosphorylethanolamine. By methylation analysis it was shown that position 6' is the attachment site for the polysaccharide moiety in lipopolysaccharide. These backbone species carry up to six molecules of ester- and amide-bound fatty acids. Four molecules of (R)-3-hydroxytetradecanoic acid are linked directly to the lipid A backbone (at positions 2, 3, 2', and 3'). Laser desorption mass spectrometry showed that both (R)-3-hydroxytetradecanoic acids linked to the non-reducing sugar unit carry, at their 3-hydroxyl group, either two molecules of hexadecanoic acid or one molecule of tetradecanoic and one of hexadecanoic acid. It also suggested that the (R)-3-(tetradecanoyloxy)-tetradecanoic acid was attached at position 2', whereas (R)-3-(hexadecanoyloxy)-tetradecanoic acid was attached at position 3', or at positions 2' and 3'. Therefore, the occurrence of three backbone disaccharides differing in amino sugar composition and presence of a hybrid disaccharide differentiate the lipid A of this C. jejuni strain from enterobacterial and other lipids A described previously.     

Structural studies of lipid A from Pseudomonas aeruginosa PAO1: occurrence of 4-amino-4-deoxyarabinose.

Lipid A derived from Pseudomonas aeruginosa PAO1 contains a biphosphorylated 1-6-linked glucosamine disaccharide backbone. The reducing glucosamine has an unsubstituted glycosidically linked phosphate at C-1. The nonreducing glucosamine has an ester-bound phosphate at C-4' which is nonstoichiometrically substituted with 4-amino-4-deoxyarabinose. Induction of 4-amino-4-deoxyarabinose was dependent on cultural conditions. No pyrophosphate groups were detected. Acyloxyacyl diesters are formed by esterification of the amide-bound 3-hydroxydodecanoic acid with dodecanoic acid and 2-hydroxydodecanoic acids in an approximate molar ratio of 2:1. Dodecanoic and 3-hydroxydecanoic acids are esterified to positions C-3 and C-3' in the sugar backbone. All hydroxyl groups of the glucosamine disaccharide except C-4 and C-6' are substituted. Lipopolysaccharide chemical analyses measured glucose, rhamnose, heptose, galactosamine, alanine, phosphate, and glucosamine. The proposed lipid A structure differs from previous models. There are significant differences in acyloxyacyl diesters, and the proposed model includes an aminopentose substituent.     

Structural characterization of the lipid A component of Pseudomonas aeruginosa wild-type and rough mutant lipopolysaccharides

The structure of the lipid A component of lipopolysaccharides isolated from two wild-type strains (Fisher 2 and 7) and one rough mutant (PAC 605) of Pseudomonas aeruginosa was investigated using chemical analysis, methylation analysis, combined gas-liquid chromatography/mass spectrometry, laser-desorption mass spectrometry and NMR spectroscopy. The lipid A backbone was found to consist of a pyranosidic beta 1,6-linked D-glucosamine disaccharide [beta-D-GlcpN-(1----6)-D-GlcpN], phosphorylated in positions 4' and 1. Position 6' of the beta-D-GlcpN-(1----6)-D-GlcpN disaccharide was identified as the attachment site of the core oligosaccharide and the hydroxyl group at C-4 was not substituted. Lipid A of the three P. aeruginosa strains expressed heterogeneity with regard to the degree of acylation: a hexaacyl as well as a pentaacyl component were structurally characterized. The hexaacyl lipid A contains two amide-bound 3-O-acylated (R)-3-hydroxydodecanoic acid groups [12:0(3-OH)] at positions 2 and 2' of the GlcN dissacharide and two ester-bound (R)-3-hydroxydecanoic acid groups [10:0(3-OH)] at positions 3 and 3'. The pentaacyl species, which represents the major lipid A component, lacks one 10:0(3-OH) residue, the hydroxyl group in position 3 of the reducing GlcN residue being free. In both hexa- and pentaacyl lipid A the 3-hydroxyl group of the two amide-linked 12:0(3-OH) residues are acylated by either dodecanoic (12:0) or (S)-2-hydroxydodecanoic acid [12:0(2-OH)], the lipid A species with two 12:0(2-OH) residues, however, being absent. The presence of only five acyl residues in the major lipid A fraction may account for the low endotoxic activity observed with P. aeruginosa lipopolysaccharide.     

Lipid A mutants of Salmonella typhimurium. Purification and characterization of a lipid A precursor produced by a mutant in 3-deoxy-D-mannooctulosonate-8-phosphate synthetase.

We describe here the isolation, purification, and structural characterization of a lipid A precursor synthesized under nonpermissive conditions by a mutant of Salmonella typhimurium conditionally defective in the synthesis of the 3-deoxy-D-mannoctulosonate (2-keto-3-deoxyoctonate, KDO) region of the lipopolysaccharide. The precursor was isolated free from lipopolysaccharide, murein, and phospholipids by extraction of delipidated cells with 90% phenol/CHCL3/petroleum ether. The molecule was recovered from the phenol phase after precipitation of lipopolysaccharide with H2O and subsequently purified by DEAE-cellulose chromatography. Structural analyses showed that the lipid A precursor is a phosphorylated glucosamine disaccharide containing one ester and two amide-linked residues of beta-hydroxymyristate. In contrast to lipid A, the precursor disaccharide lacks ester-linked 12:0 and 14:0 fatty acids as well as KDO. The molecule contains 2 phosphate residues both of which were identified as phosphomonoesters by 31P NMR spectroscopy. One of the phosphomonoesters is located in position 1 of the reducing terminal glucosamine residue; the location of the other phosphomonoester was not determined. The structure of the precursor provides strong support for the conclusion that KDO incorporation occurs at an early stage in lipid A biosynthesis prior to the incorporation of ester-linked saturated fatty acids.     

Structure of a fucoidan from the brown seaweed Fucus serratus L

A fucoidan consisting of L-fucose, sulfate and acetate in a molar proportion of 1:1:0.1 and small amounts of xylose and galactose were isolated from the brown seaweed Fucus serratus L. The fucoidan structure was investigated by 1D and 2D 1H and 13C NMR spectroscopy of its desulfated and de-O-acetylated derivatives as well as by methylation analysis of the native and desulfated polysaccharides. A branched structure was suggested for the fucoidan with a backbone of alternating 3- and 4-linked alpha-L-fucopyranose residues, -->3)-alpha-L-Fucp-(1-->4)-alpha-L-Fucp-(1-->, about half of the 3-linked residues being substituted at C-4 by trifucoside units alpha-L-Fucp-(1-->4)-alpha-L-Fucp-(1-->3)-alpha-L-Fucp-(1-->. Minor chains built up of 4-linked alpha-fucopyranose and beta-xylose residues were also detected, but their location, as well as the position of galactose residues, remained unknown. Sulfate groups were shown to occupy mainly C-2 and sometimes C-4, although 3,4-diglycosylated and some terminal fucose residues may be nonsulfated. Acetate was found to occupy C-4 of 3-linked Fuc and C-3 of 4-linked Fuc in a ratio of about 7:3.     

Structural studies on the D-arabinose-containing lipid A from Rhodospirillum tenue 2761

Structural studies carried out on the isolated free lipid A of Rhodospirillum tenue 2761 revealed a new type of structure for this lipid. The lipid A backbone of 1',6-linked glucosamine disaccharide (central disaccharide) is substituted by three different sugar residues: the non-reducing end of the disaccharide by 4-amino-4-deoxy-L-arabinose 1-phosphate and its reducing end glycosidically by D-arabinofuranose 1-phosphate; further, the reducing glucosamine of the disaccharide is branched to a third glucosamine residue by a 1',4-glycosidic linkage. The amino and the hydroxyl groups of the central disaccharide are acylated by 3-hydroxydecanoic acid (amide-linked) and palmitic and myristic acids (ester-linked). Neither amino nor hydroxyl groups of the three external sugar residues are acylated. The results suggest the following chemical structure for the lipid A of R. tenue 2761: (formula: see text).     

Chemical structure of the lipopolysaccharide of Haemophilus influenzae strain I-69 Rd-/b+. Description of a novel deep-rough chemotype

The chemical structure of the lipopolysaccharide of a deep-rough mutant (strain I-69 Rd-/b+) of Haemophilus influenzae was investigated. The hydrophilic backbone of lipid A was shown to consist of a beta-(1',6)-linked D-glucosamine disaccharide with phosphate groups at C-1 of the reducing D-glucosamine and at C-4' of the non-reducing one. Four molecules of (R)-3-hydroxytetradecanoic acid were found directly linked to the lipid A backbone, two by amide and two by ester linkage (positions 2,2' and 3,3', respectively). Laser-desorption mass spectrometry showed that both 3-hydroxytetradecanoic acids linked to the non-reducing glucosamine carry tetradecanoic acid at their 3-hydroxyl group, so that altogether six molecules of fatty acid are present in lipid A. The lipopolysaccharide was the first described to contain only one sugar unit linked to lipid A. This, sugar in accordance with a previous report [Zamze et al. (1987) Biochem. J. 245, 583-587], was shown to be a dOclA phosphate. The phosphate group was found at position 4, but the analytical procedures employed (permethylation and methanolysis followed by gas-liquid chromatography/mass spectrometry) also revealed dOclA 5-phosphate. Since a cyclic 4,5-phosphate could be ruled out by 31P-NMR, we conclude that, in this lipopolysaccharide, a mixture of dOclA 4- and 5-phosphate is present. By methylation analysis of the dephosphorylated, deacylated and reduced lipopolysaccharide the attachment site of the dOclA was assigned to position C-6' of the non-reducing glucosamine of lipid A. The anomeric linkages present in the lipopolysaccharide were assessed by 1H-NMR and 13C-NMR of deacylated lipopolysaccharide. The saccharide backbone of this Haemophilus influenzae lipopolysaccharide possesses the following structure: (Formula; see text)     

Characterization of lipid A from Pseudomonas aeruginosa O-antigenic B band lipopolysaccharide by 1D and 2D NMR and mass spectral analysis.

The Lipid A from the lipopolysaccharide of Pseudomonas aeruginosa was examined by high-field nuclear magnetic resonance spectroscopy (NMR) and mass spectrometry (MS). The backbone structure and the position of phosphate substituents were unambiguously established by one- and two-dimensional 1H, 13C, and 31P NMR techniques on a de-O-acylated Lipid A sample. The Lipid A has a beta-(1,6)-glucosamine disaccharide structure which is substituted by phosphomonoesters through glycosidic bonds at C-1 and at C-4'. The configuration of the glycosidically linked phosphate at position C-1 was identified as alpha which is the same as that of Enterobacterial Lipid A. Chemical analysis revealed that the Lipid A contained 2-hydroxydodecanoic, 3-hydroxydodecanoic, dodecanoic, 3-hydroxydecanoic, and hexadecanoic acids in the approximate molar ratios 2.2:2.0:0.2:0.8:0.4. From 1H NMR and fast atom bombardment (FAB) mass spectrometry on the de-O-acylated Lipid A, it was established that both glucosamine residues were N-acylated by 3-hydroxydodecanoic acid. The identity and positions of the ester bound fatty acids in the intact Lipid A were investigated by negative ion FAB-MS. In addition to the hexaacyl and pentaacyl Lipid A species, a tetraacyl species was identified. Heterogeneity due to hydroxylated and nonhydroxylated dodecanoic acid esters could be uniquely localized to the nonreducing beta-glucosamine residue from the fragmentation pattern observed in the negative ion FAB-MS. The complete structure of the Lipid A from P. aeruginosa will be useful in understanding the determinants responsible for the endotoxin activity of this molecule.     

Characterization of lipid A and polysaccharide moieties of the lipopolysaccharides from Vibrio cholerae.

Lipid A and polysaccharide moieties obtained by mild acid hydrolysis of the lipopolysaccharides from Vibrio cholerae 569 B (Inaba) and Vibrio el-tor (Inaba) were characterized. Heterogeneity of lipid A fractions was indicated by t.l.c. and by gel filtration of the de-O-acylated products from mild alkaline methanolysis of the lipids. Presumably lipid A contains a glucosamine backbone, and the fatty acids are probably bound to the hydroxyl and amino groups of glucosamine residues. Approximately equal amounts of fatty acids C16:0, C18:1 and 3-hydroxylauric acid were involved in ester linkages, but 3-hydroxymyristic acid was the only amide-linked fatty acid. Sephadex chromatography of the polysaccharide moiety showed the presence of a high-molecular-weight heptose-free fraction and a low-molecular-weight heptose-containing fraction. Haemagglutination-inhibition assays of these fractions showed the heptose-free fraction to be an O-specific side-chain polysaccharide, whereas the heptose-containing fraction was the core polysaccharide region of the lipopolysaccharides. Identical results were obtained for both organisms.     

Structural heterogeneity regarding local Shwartzman activity of lipid A

The relation of chemical structure to local Shwartzman activity of lipid A preparations purified by thin-layer chromatography from five bacterial strains was examined. Two lipid A fractions from E. coli F515--Ec-A2 and Ec-A3--exhibited strong activity, similar to that of previous synthetic E. coli-type lipid A (compound 506 or LA-15-PP). The Ec-A3 fraction contained a component that appeared to be structurally identical to compound 506, and the main component of Ec-A2 fraction was structurally similar to compound 506 except that it carried a 3-hydroxytetradecanoyl group at the C-3' position of the backbone in place of a 3-tetradecanoyloxytetradecanoyl group. Free lipid A (12 C) and purified lipid A fractions, Ec-A2 (12 C) and Ec-A3 (12 C), respectively, obtained from bacteria grown at 12 C, exhibited activity comparable to Ec-A2 or Ec-A3. In these preparations, a large part of the 3-dodecanoyloxytetradecanoyl group might be replaced by 3-hexadecenoyloxytetradecanoyl group. Salmonella minnesota R595 free lipid A also contained at least two active lipid A components as seen in E. coli lipid A, but the third component corresponding to the synthetic Salmonella-type lipid A (compound 516 or LA-16-PP) exhibited low activity. A lipid A fraction, Cv-A4 from Chromobacterium violaceum IFO 12614, which was proposed to have two acyloxyacyl groups at the C-2 and C-2' positions with other acyl groups, exhibited weaker activity than the free lipid A or LPS. The purified lipid A fractions from Pseudomonas diminuta JCM 2788 and Pseudomonas vesicularis JCM 1477 contained an unusual backbone with 2,3-diamino-2,3-dideoxy-D-glucose disaccharide phosphomonoester, and these lipid A (Pd-A3 and Pv-A3) exhibited strong activity comparable to the E. coli lipid A. Thus, the present results show that the local Shwartzman reaction can be expressed by partly different lipid A structures in both hydrophilic backbone and fatty acyl residues; when they have the same backbone the potency varies markedly depending on the structure of the acyl residues.